Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and $37^{circ}C$ than in 2.5% and $25^{circ}C$. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and $25^{circ}C$) was changed into that reflecting human body (0.9% salinity and $37^{circ}C$), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at $37^{circ}C$. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose-dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.